1. Field of the Invention
The invention relates to synthetic lipid vesicles and the method of their manufacture, encapsulating biologically active materials and to their use.
2. Brief Description of the Prior Art
Prior to our invention, several methods have been available to make synthetic liposomes, encapsulating biologically active materials. For example, Robinson, Trans. Faraday Soc., 56:1260-1264 (1960) and Papahadjopoulos et al. (Biochim. Biophys. Acta, 135, 639, 1967) described a method of forming phospholipid dispersions from an ether-lipid-aqueous two-phase system that involved the evaporation of the ether by bubbling nitrogen through the mixture. There was no attempt to use this procedure to entrap organic materials and the trapping efficiency was not investigated in detail. A similar evaporation technique from a chloroformaqueous two-phase system was described by Chowhan et al., in Biochim. Biophys. Acta, 266:320-342 (1972 ). This procedure also involved the use of excess aqueous phase and the slow removal of the chloroform phase in order to produce a uniform population of phospholipid vesicles. There was no attempt to maximize captured aqueous material and no investigation into the trapping efficiency of this procedure.
Bangham et al. in J. Mol. Biol., 13:238-252 (1965) described multilamellar lipid vesicles which could be characterized as having a small trapping volume, a low trapping efficiency (of 10 percent) and a confined aqueous space (15 to 35 A).
The small unilamellar vesicles produced by ultrasonication, described initially by D. Papahadjopoulos and N. Miller (Biochim. Biophys. Acta, 135:624-638 [1967]) and by many others since then, have very low capture efficiencies and are unsuitable for encapsulating large macromolecules due to their small aqueous compartment (250 A).
Lipid vesicles prepared by injection of the lipids in an organic phase into an aqueous solution were described by Batzri and Korn (Biochim. Biophys. Acta, 298:1015 [1973]) using ethanol and by Deamer and Bangham in Biochim. Biophys. Acta, 443:629-634 (1976) using ether. These methods produce unilamellar or paucilamellar vesicles but, again do not achieve high efficiencies in encapsulation. In the case of the ethanol injection this low efficiency is due to the large aqueous volume in which the ethanol is dispersed, and the small size of the vesicles produced by the technique. In the case of the ether injection technique, it is due to a combination of the large volume of aqueous space the ether is injected into, the small amounts of lipid employed in the method, and the manner in which the vesicles form.
A preparation of large unilamellar vesicles has been described by Papahadjopoulos et al. in Biochim. Biophys. Acta, 394:483-491 (1975) that involves a unique calcium-induced structural change in the lipid vesicle, but this technique is restricted to a single phospholipid (phosphatidylserine) and also has a relatively low efficiency for encapsulation due to the method of reconstitution of the vesicles.
Another lipid vesicle preparation has been described in German Pat. No. 2,532,317, which involves centrifugation of a lipid-water-ether emulsion into an aqueous phase. The disadvantage of this technique is that high speed centrifugation is required and a large amount of the lipid-aqueous emulsion becomes trapped at the interface and does not enter the aqueous phase. This reduces the percentage of material entrapment.
The U.S. Pat. No. 3,804,776 is noteworthy for its disclosure of a method for producing oil and fat encapsulated amino acids or polypeptides by dispersing powders of the desired material for encapsulation in a molten mixture of the fat or oil and thereafter pouring the molten mixture into water. The encapsulated material is contained within relatively large droplets of lipid which restricts their use to oral administration to an animal. The method is somewhat restrictive to that it apparently is limited to encapsulation of powders, and the lipid does not form a bilayer.
Finally, mention may be made of U.S. Pat. No. 4,016,100 which describes the entrapment of certain pharmaceuticals in lipid vesicles by freezing the aqueous phospholipid dispersion of pharmaceutical and lipid. The pharmaceutical compounds disclosed for encapsulation by the reference method generally exhibit a high partition coefficient into an organic phase from water. Therefore it would be expected that the material for encapsulation would penetrate into the phospholipid bilayers of the product vesicles. Theoretically this would provide a high degree of encapsulation but there remains an open question as to the bio-availability of the total material encapsulated. It would also be expected that relatively high rates of encapsulation would not be obtained if the technique were applied to encapsulate pharmaceuticals which are of a more polar nature and less likely to penetrate the vesicle bilayers.
By the method of our invention, oligolamellar lipid vesicles (synthetic liposomes) may be constructed rapidly, conveniently, under mild conditions, in high yields, and in such a manner that they incorporate a high percentage of a wide variety of biologically active material processed with them. Representative of material which may be encapsulated by the method of the invention are pharmaceutically active compounds and compositions thereof, carbohydrates, nucleotides, polynucleotides (both naturally occurring and synthetic) pesticides, including fungicides, insecticides, miticides, nematocides and mollusicides, water soluble fertilizers and agricultural nutrients, peptides, proteins, enzymes, viruses and the like. Many of these materials do not normally penetrate the plasma membrane of cells and may be inactivated in circulation within a living organism or by contact with tissue and organ cultures. In the case of pesticides and agricultural nutrients or fertilizers they may be removed from the area of application by rain or irrigation. Encapsulation of such materials protects them from inactivation or removal, i.e.; maintains bioavailability. Bacterial cells such as C. parvum and E. coli and the like may also be encapsulated by the method of the invention for protection and bioavailability.
The method of the invention may also be used to encapsulate cosmetic preparations which may be usefully employed as described in U.S. Pat. No. 3,957,971.